Studies suggesting endocrine disruption in wildlife are extensive, but detailed mechanistic data are lacking. Nonetheless, there is considerable concern that low level exposure to mixtures of endocrine disrupting chemicals (EDC) is a risk for both human and wildlife health. Of particular concern are early developmental stages, which are most sensitive to interference of genetic programming by EDC and which can affect whole organism fitness, with potential population level effects. In this continuing study of endocrine disruption in a sentinel species, we are evaluating the effects of putative EDC, associated with the Massachusetts Military Reservation (MMR) Superfund site on Cape Cod, on reproduction in the fresh water turtle, Chrysemys picta. Developmental parameters to be evaluated include gonadal development and growth (oogenesis, oocyte number and size, vitellogenin, spermatogenesis, spermatogonial and Sertoli cell number, germ cell apoptosis) and function (epidydymal growth, sex dimorphic characteristics, steroid and aryl hydrocarbon receptor activity and P450aromatase induction). Specific Aims are: 1) to compare parameters of gonadal growth, steroid receptor function, and AhR expression during development in neonatal, juvenile, and adult turtles from control and contaminated test sites and, in "cross-over" experiments, in animals from each site maintained in conditions simulating the respective natural environments; 2) to isolate the major components of the xenobiotic mixtures present in surface water and pond sediment and in turtle egg yolk and gonadal tissue extracts by HPLC and GC/MS analysis; 3) to demonstrate on a molecular level a cause-and-effect relationship between effects identified in Aim 1 and chemicals identified in Aim 2. These latter studies will employ a novel zebrafish bioassay for identification of bioactive chemicals and their effects on several gene products, followed by evaluation of chemical effects on adult and developing turtles. Particular emphasis will be placed on the combined effects of estrogen-like and dioxin-like compounds on the levels of wildtype and splice variant P450aromatase mRNAs. All of these studies will exploit our active collaborations with investigators on Projects 5, 6, 7, and 9.